US20080104725A1 - Methods For The Modulation of Oleosin Expression In Plants - Google Patents

Methods For The Modulation of Oleosin Expression In Plants Download PDF

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US20080104725A1
US20080104725A1 US11/576,389 US57638905A US2008104725A1 US 20080104725 A1 US20080104725 A1 US 20080104725A1 US 57638905 A US57638905 A US 57638905A US 2008104725 A1 US2008104725 A1 US 2008104725A1
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nucleic acid
acid sequence
oleosin
plant
seed
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Rodrigo M. Siloto
Maurice M. Moloney
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UTI LP
SemBioSys Genetics Inc
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SemBioSys Genetics Inc
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23D—EDIBLE OILS OR FATS, e.g. MARGARINES, SHORTENINGS, COOKING OILS
    • A23D9/00—Other edible oils or fats, e.g. shortenings, cooking oils
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis

Definitions

  • the present invention relates to plant genetic engineering methods. More specifically, the present invention relates to methods to modulate the expression levels of oleosin proteins in plants.
  • Plant seeds represent an important source of nutrients for both human and animal use.
  • plant seed proteins represent a major component of animal feed and plant seed oil is used for the production of vegetable oil which is used extensively for human consumption.
  • oil bodies In seeds, the water insoluble oil fraction is stored in discrete subcellular structures variously known in the art as oil bodies, oleosomes, lipid bodies or spherosomes (Huang, 1992 Ann. Rev. Plant Mol. Biol. 43: 177-200), having a diameter ranging between 0.5 and 2.0 micrometers (Tzen, 1993 Plant Physiol. 101: 267-276).
  • oil bodies Besides a mixture of oils (triacylglycerides), which chemically are defined as glycerol esters of fatty acids, oil bodies comprise phospholipids and a number of associated proteins, collectively termed oil body proteins.
  • oil bodies are considered to be a triacylglyceride matrix encapsulated by a monolayer of phospholipids in which oil body proteins are embedded (Huang, 1992 Ann. Rev. Plant Mol. Biol. 43: 177-200).
  • the seed oil present in the oil body fraction of plant species is a mixture of various triacylglycerides, of which the exact composition depends on the plant species from which the oil is derived.
  • SM — 3-29875 contains a DNA insertion in the second exon of Atol1 (Tissier A. F. et al., Plant Cell 11: 1841-1852) and SALK — 072403 contains a single insertion in Atol2 (Alonso J. M. et al., Science 301: 653-657).
  • the present invention relates to methods for preparing seed derived products from seed, in which the composition of seed storage reserves, notably the seed lipid and protein contents, have been altered.
  • the present invention provides methods for preparing seed derived products from seed, in which the seed reserves have been altered by modulation of oleosin gene expression and more particularly the suppression of oleosin gene expression.
  • the present invention provides a method for preparing a plant seed derived product from plants seeds comprising:
  • the present invention also provides a method to increase the protein content in plants seeds, comprising:
  • the present invention also provides a method to decrease the lipid content in plants seeds, comprising:
  • the seed obtained from these plants prepared in accordance with the present invention may be used as a source for the preparation of a variety of plant seed derived products.
  • the present invention provides a method to suppress expression of an oleosin protein in a plant comprising:
  • the nucleic acid sequence which upon transcription generates a RNA nucleic acid sequence that is complementary to a nucleic acid sequence encoding an oleosin mRNA is linked to a nucleic acid sequence encoding an oleosin. Accordingly, the present invention provides a method to suppress expression of an oleosin protein in a plant comprising:
  • the nucleic acid sequence capable of controlling expression in plant cells permits expression in plant seed cells and the transformed plant is a plant capable of setting seed.
  • the promoter is a seed-preferred promoter.
  • the present invention provides a method to suppress expression of an oleosin protein in the seed of a plant comprising:
  • the present invention provides a method to suppress expression of an oleosin protein in the seed of a plant comprising:
  • the present invention provides a method for preparing a plant seed derived product from plant seeds comprising:
  • the chimeric nucleic acid construct is introduced into the plant cell under nuclear genomic integration conditions. Under such conditions the chimeric nucleic acid sequence is stably integrated in the plant's genome.
  • FIG. 1 Structure of RNA molecules that trigger post-transcriptional gene silencing.
  • the molecule contain a self complementary region composed by a portion identical to the target mRNA (sense portion) and another portion complementary identical to the target mRNA (antisense portion).
  • the hairpin RNA might contain the sense portion in the 3′ end and antisense portion in the 5′ end (left panel) or the antisense portion in the 3′ end and the sense portion in the 5′ end (right panel).
  • the molecule contain a self complementary region composed by a portion identical to the target mRNA (sense portion), a region that is not identical or complementary to the target mRNA (loop) and another portion complementary to the target mRNA (antisense portion).
  • the hairpin-loop RNA might contain the sense portion in the 3′ end followed by the loop portion, followed by the antisense portion in the 5′ end (left panel) or the antisense portion in the 3′ end, followed by the loop portion, followed by the sense portion in the 5′ end (right panel).
  • FIG. 2 Different configuration of cassettes used to suppress one or multiple oleosin genes.
  • Hairpin cassette to suppress a single oleosin gene a promoter fragment is fused to an oleosin coding region, followed by the same coding region in inverted orientation followed by a terminator fragment.
  • hairpin cassette a promoter fragment is fused to an oleosin coding region in inverted orientation, followed by the same coding region in the upright orientation followed by a terminator fragment.
  • Hairpin-loop cassette to suppress a single oleosin gene a promoter fragment is fused to an oleosin coding region, followed by a DNA fragment that is not related to the coding region (for example, an intron), followed by the same coding region in inverted orientation followed by a terminator fragment.
  • Hairpin-loop cassette to suppress a single oleosin gene a promoter fragment is fused to an oleosin coding region inverted orientation, followed by an unrelated DNA fragment, followed by the same coding region in correct orientation, followed by a terminator fragment.
  • Hairpin-loop cassette to suppress three different oleosin genes a promoter fragment is fused to three oleosin coding regions in tandem, followed by an unrelated DNA fragment, followed by the same three coding regions in the same order in inverse orientation, followed by a terminator fragment.
  • Hairpin-loop cassette to suppress three different oleosin genes a promoter fragment is fused to the first oleosin coding regions in correct orientation, followed by the second oleosin coding region in inverse orientation, followed by the third oleosin coding region in correct orientation followed by an unrelated DNA fragment, followed by third coding region in inverse orientation, followed by the second coding region in correct orientation, followed by the first coding region I inverse orientation, followed by a terminator fragment.
  • FIG. 3 Scheme for construction of antisense and hairpin cassettes.
  • the cDNA encoding for the 18 kDa oleosins from Arabidopsis thaliana (Atol1) (a) was obtained by PCR reaction with the primers NTD and CTR and inserted in the plasmid pSBS2090 (b) previously digested with the enzyme SwaI between the phaseolin promoter and terminator.
  • the insertion provided the plasmid pAntisense (c) and pHairpin (d) that were selected according to the profile obtained with NcoI and HindIII/SalI.
  • FIG. 4 Scheme for construction of hairpin-loop cassette.
  • the plasmid pHairpin (b) was digested with the enzymes KpnI and BamHI.
  • the hairpin cassette was sub cloned in the vector pUC19 (a), generating the plasmid pUC-Hairpin (c).
  • a fragment corresponding for the intron of Atol1 gene was amplified using the primers IntronD and Intron R (d). These primers created a restriction site for SpeI in each end.
  • the fragment was purified, digested with SpeI and inserted in the plasmid pUC-Hairpin.
  • the resulting plasmid is called pHairpin-intron (e).
  • FIG. 5 Scheme for construction of binary vectors carrying suppression cassettes.
  • the plasmids pAntisense, pHairpin and pHairpin+intron (a) were digested with BamHI and KpnI.
  • the cassettes were inserted in the binary vector pSBS3000 (b).
  • the resulting plasmids were respectively called pSBS3000 Antisense, pSBS3000 Hairpin and pSBS3000 Hairpin+intron (c).
  • FIG. 6 Suppression of Atol1 oleosins in seeds from transgenic Arabidopsis lines. Oil bodies are extracted from seeds of Arabidopsis lines containing the suppression cassettes (lane 3) antisense, (lane 2) harpin (lane 1) harpin+intron. Oil body associated proteins are loaded in a SDS-PAGE 15% and stained with Comassie blue R250.
  • FIG. 7 In vivo comparison of oil bodies size in Arabidopsis lines.
  • Panel “a” shows the oil bodies from wildtype (untransformed) Arabidopsis seeds.
  • Panel “b” shows the oil bodies from Arabidopsis seeds containing the antisense suppression cassette.
  • Panel “c” shows the oil bodies from Arabidopsis seeds containing the hairpin suppression cassette.
  • Panel “d” shows the oil bodies from Arabidopsis seeds containing the hairpin+intron suppression cassette.
  • the red circles represent oil bodies.
  • the white bars indicate reference distances in “ ⁇ m”.
  • FIG. 8 In vitro comparison of oil bodies size in Arabidopsis lines.
  • Panel “A” shows the oil bodies from wildtype (untransformed) Arabidopsis seeds.
  • Panel “B” shows the oil bodies from Arabidopsis seeds containing the suppression cassette hairpin+intron. The material stained in blue corresponds predominantly of protein bodies. The open circles represent oil bodies.
  • Panel “C” shows the oil bodies from wild type like (null) Arabidopsis line segregated from plants containing the suppression cassette hairpin+intron.
  • FIG. 9 Thin layer chromatography of oil body-lipids. Oil bodies were isolated from different plants and total lipids were extracted from these organelles. Lipids were applied on silica Gel 60 F254 plates and half-developed with chloroform-methanol-acetic acid-formic acid-water (70:30:12:4:2 [v/v]) and fully developed with hexane-diethyl ether-acetic acid (65:35:2 [v/v]) according to Vance and Russell (1990). The lipids were visualized by heating the plates after they have been dipped in a solution containing cupric acetate (3%) and phosphoric acid (8%).
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • TAG triacylglycerol
  • FIG. 10 The introduction of a recombinant oleosin to rescue the phenotype are showed in left panels and confocal section in right panels.
  • SupAtol1-Loop (Hairpin-Loop) plant Left panel: SDS-PAGE profile of oil body associated proteins. Atol1 polypeptide is indicated by the black arrow. Right panel: confocal section of mature embryo. White arrows show large oil bodies.
  • FIG. 11 Comparison of germination of wild-type and SupAtol1-Loop plants in different conditions. The germination rate in each batch was scored by visualisation of radicle emergence every 24 hours.
  • A Wet filter paper; Light;
  • B Wet filter paper; stratified seeds;
  • D Half strength MS media+Sucrose; Light;
  • E Half strength MS media ⁇ Sucrose; Dark;
  • F Half strength MS media+Sucrose; Dark.
  • FIG. 12 Fate of oil bodies after germination and seedling development.
  • A and (B) Confocal sections of wild-type Arabidopsis seedlings after 2 and 4 days after imbibition respectively. Oilbodies were stained with Nile red.
  • C (D) and (E) Confocal sections of oleosin suppressed Arabidopsis seedlings after 2 and 4 and 6 days after imbibition respectively. Oilbodies were stained with Nile red.
  • nucleic acid construct and “nucleic acid sequence” as used herein refers to a polynucleoside or polynucleotide consisting of monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The terms also include modified or substituted sequences comprising non-naturally occurring monomers or portions thereof.
  • the nucleic acid constructs of the present invention may be deoxyribonucleic acid constructs (DNA) or ribonucleic acid constructs (e.g. RNA, mRNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil.
  • the constructs may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine.
  • chimeric refers to at least two linked nucleic acid sequences which are not naturally linked.
  • a nucleic acid sequence constituting a plant promoter linked to a nucleic acid sequence encoding an mRNA complementary to a nucleic acid sequence encoding an oleosin is a chimeric nucleic acid sequence.
  • nucleic acid sequences are capable of hybridizing under at least moderately stringent hybridization conditions to form a nucleic acid duplex.
  • At least moderately stringent hybridization conditions it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length.
  • T m the stability of a nucleic acid duplex, or hybrids
  • T m the sodium ion concentration and temperature
  • T m 81.5° C. ⁇ 16.6 (Log 10 , [Na + ])+0.41(% (G+C) ⁇ 600/1), or similar equation).
  • the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature.
  • a 1% mismatch may be assumed to result in about a 1° C.
  • stringent hybridization conditions are selected.
  • the following conditions may be employed to achieve stringent hybridization: hybridization at 5 ⁇ sodium chloride/sodium citrate (SSC)/5 ⁇ Denhardt's solution/1.0% SDS at T m ⁇ 5° C. based on the above equation, followed by a wash of 0.2 ⁇ SSC/0.1% SDS at 60° C.
  • Moderately stringent hybridization conditions include a washing step in 3 ⁇ SSC at 42° C.
  • mRNA or “messenger RNA” as used herein refers to a polynucleotide which is the product of transcription of a DNA sequence and capable of being translated into a polypeptide.
  • oil body or “oil bodies” as used herein refers to any oil or fat storage organelle in plant cell (described in for example: Huang (1992) Ann. Rev. Plant Mol. Biol. 43: 177-200).
  • oleosin and “oleosin polypeptides” as may be used herein interchangeably refer to any and all oleosin polypeptides, including the oleosin polypeptides listed in Table 1 (SEQ ID NO:1 to 84), as well as a polypeptide molecule which (i) is substantially identical to the amino acid sequences constituting any oleosin polypeptides set forth herein or (ii) is encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding oleosin but for the use of synonymous codons.
  • the oleosin polypeptide is preferably from plant origin.
  • substantially identical it is meant that two polypeptide sequences preferably are at least 75% identical, and more preferably are at least 85% identical and most preferably at least 95% identical, for example 96%, 97%, 98% or 99% identical.
  • amino acid sequences of such two sequences are aligned, preferably using the Clustal W algorithm (Thompson, J D, Higgins D G, Gibson T J, 1994, Nucleic Acids Res. 22 (22): 4673-4680, together with BLOSUM 62 scoring matrix (Henikoff S. and Henikoff J. G., 1992, Proc. Natl. Acad. Sci.
  • hairpin or “hairpin structure” as used herein refers to an RNA duplex structure formed by the hybridization of a first and second portion of an mRNA polynucleotide wherein the first portion of the mRNA polynucleotide is located immediately 5′ relative to the second portion of the mRNA polynucleotide (See: FIG. 1 b ).
  • the “hairpin” can also further comprise 3′ and/or 5′ single-stranded region(s) extending from the double-stranded stem segment.
  • polynucleotide loop or “loop” as used herein refers to one or more mRNA nucleotides separating the nucleic acid sequence encoding an RNA polynucleotide complementary to a nucleic acid sequence encoding an oleosin from the nucleic acid sequence capable of encoding an oleosin (See: FIG. 1 c ).
  • the polynucleotide loop can be any intervening sequence.
  • the polynucleotide loop has no secondary structure.
  • modified oleosin profile means that the plant has steady-state oleosin levels that are reduced as compared to non-transformed plants.
  • seeds with a modified oleosin profile also have an increase in total protein content and a decrease in lipid content as compared to a non-transformed seed.
  • the “modified oleosin profile” is preferably a reduction in the steady-state levels of specific oleosin proteins as compared to the same proteins from non-transformed plants. For the purpose of this application, this reduction results after the introduction of the chimeric nucleic acid sequence into the plant cell and regeneration of a mature plant.
  • the steady-state protein levels are reduced to a level from about 10% to about 90% compared to the unaltered protein levels. More preferably, the steady-state protein levels are reduced to a level 50% to 90% compared to the unaltered protein levels and most preferably, the steady-state protein levels are reduced 80% to 90% compared to the unaltered protein levels present in plants not comprising the chimeric nucleic acid sequence of the present invention.
  • Techniques to determine the steady-state protein levels include densitometry, a quantitative Western blot analysis or the use of an ELISA. Examples of protocols can be found in Coligan et al. Current Protocols in Protein Science, vol 3.
  • the present invention provides methods to suppress the expression of the endogenously present oleosin polypeptides in plants.
  • the methods described herein are based on modifications of a plant genome with the objective of suppressing the biosynthetic production of oleosins using a chimeric nucleic acid sequence comprising a nucleic acid sequence that encodes an RNA polynucleotide complementary to a nucleic acid sequence encoding an oleosin mRNA.
  • the present invention relates to preparing seed derived products from seed, in which the composition of seed storage reserves, notably the seed lipid and protein contents, have been altered.
  • the present invention provides methods for preparing seed derived products from seed, in which the seed reserves have been altered by modulation of oleosin gene expression and more particularly the suppression of oleosin gene expression.
  • the present inventors have found that the introduction of a nucleic acid sequence which upon transcription generates a RNA nucleic acid sequence that is complementary to an oleosin mRNA results in suppression of the expression levels of endogenous plant oleosins.
  • This reduction in expression levels of oleosins results in a surprising modulation of the size of the plant oil bodies present in plant seeds, and, significantly, in a substantial alteration of the seed composition.
  • the lipid and protein contents of the seed may be modulated.
  • the methodologies herein described are further advantageous in that they permit specific modulation of the expression levels of endogenous oleosin polypeptides.
  • the seeds obtained in accordance with the present invention may be used to prepare a wide range of products for human and animal use, including in the formulation of food and feed products.
  • the present invention provides a method for preparing a plant seed derived product from plants seeds comprising:
  • the nucleic acid sequence which upon transcription generates a RNA nucleic acid sequence that is complementary to a nucleic acid sequence encoding an oleosin mRNA may be any nucleic acid sequence which upon transcription generates a RNA nucleic acid sequence that is complementary to a nucleic acid sequence encoding an oleosin mRNA or the corresponding oleosin cDNA. This sequence can be referred to as the “antisense sequence” herein.
  • the antisense sequence complementary to a nucleic acid sequence encoding an oleosin mRNA may conveniently be prepared by selecting a DNA sequence encoding an oleosin and using such selected DNA sequence to prepare a nucleic acid sequence complementary thereto.
  • DNA sequences encoding oleosins are well known to the art and generally available from a diverse number of sources.
  • DNA sequences encoding oleosins are preferably selected from a plant source. Exemplary DNA sequences that may be selected in this regard include the oleosin sequences obtainable from Arabidopsis (Van Rooijen et al (1991) Plant Mol. Bio.
  • Oleosin sequences that may be used in accordance herewith include those set forth as SEQ ID NO:1 to SEQ ID NO:84.
  • the corresponding nucleic acid sequences encoding the oleosin polypeptide can be readily identified via the Swiss Protein identifier numbers provided in Table 1.
  • nucleic acid sequences may be readily identified using techniques well known to those of skill in the art. For example, libraries, such as expression libraries may be screened, and databases containing sequence information may be screened for similar sequences. In accordance herewith other methods to identify nucleic acid sequences encoding oleosins may be used and novel sequences may be discovered and used.
  • the nucleic acid sequence complementary to the nucleic acid sequence encoding an oleosin mRNA is preferably a DNA sequence which upon introduction in the plant cell of the chimeric nucleic acid sequence and regeneration of the plant is transcribed into a complementary RNA polynucleotide.
  • the complementary DNA sequence may be prepared in a variety of ways including the generation of cDNA sequences using reverse transcription of mRNA.
  • a protocol for reverse-transcriptase PCR (RT-PCR) can be found in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.
  • the cDNA sequence does not contain any secondary structure. More preferably, the cDNA sequence also contains a poly-A tail for enhanced stability.
  • the length of the DNA sequence complementary to the nucleic acid sequence encoding an oleosin sequence may vary, provided however, that a sequence is used which upon expression of the chimeric nucleic acid sequence in the regenerated plant results in a reduction of the endogenously present levels of plant oleosins.
  • the term “suppression” as used herein describes a reduction in the steady-state levels of specific proteins. For the purpose of this application, this reduction results after the introduction of the chimeric nucleic acid sequence into the plant cell and regeneration of a mature plant.
  • the steady-state oleosin protein levels are reduced to a level from about 10% to about 90% compared to the unaltered protein levels.
  • the steady-state oleosin protein levels are reduced to a level 50% to 90% compared to the unaltered protein levels and most preferably, the steady-state oleosin protein levels are reduced 80% to 90% compared to the unaltered protein levels present in plants not comprising the chimeric nucleic acid sequence of the present invention.
  • Techniques to determine the steady-state protein levels include densitometry, a quantitative Western blot analysis or the use of an ELISA. Examples of protocols can be found in Coligan et al. Current Protocols in Protein Science, vol 3. The techniques listed above may be performed on either a total seed extract or on the oil body fraction.
  • the DNA sequence complementary to the nucleic acid sequence encoding an oleosin is the same length as the DNA sequence encoding the oleosin (see FIG. 2 a ) and the percentage sequence identity relative to the DNA sequence complementary to the sequence encoding the oleosin is 100%, however shorter fragments, complementary to only a portion of the sequence encoding an oleosin may also be used and the percentage identity may be lower for example 99%, 98,%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90%. Where shorter fragments are used, such fragments may for example be 95%, 90%, 85%, 80% or 75% of the length of the entire oleosin nucleotide sequence.
  • the nucleic acid sequence encoding a RNA polynucleotide complementary to a nucleic acid sequence encoding an oleosin mRNA is selected from SEQ ID NO:85 and 86.
  • the chimeric nucleic acid sequence further includes a nucleic acid sequence capable of encoding an oleosin.
  • the nucleic acid encoding an oleosin can be any nucleic acid sequence that encodes an oleosin or oleosin polypeptide as defined herein. This nucleic acid sequence can also be referred to as the “sense sequence” herein.
  • the present invention provides a method to suppress expression of an oleosin protein in a plant comprising:
  • the present invention provides a method for preparing a plant seed derived product from plant seeds comprising:
  • RNA nucleic acid sequence that is complementary to a nucleic acid sequence encoding an oleosin which in accordance herewith has been linked to a nucleic acid sequence encoding an oleosin or a fragment thereof
  • a single stranded mRNA is synthesized and that upon synthesis, due to the complementarity of the nucleic acid sequences (ii) and (iii) such mRNA will form a duplex structure.
  • a duplex structure known as a hairpin is formed.
  • the term “hairpin” has been defined previously herein and is shown schematically in FIG. 1 b.
  • the nucleic acid sequence which upon transcription generates a RNA nucleic acid sequence that is complementary to a nucleic acid sequence encoding an oleosin is complementary to the full length oleosin nucleotide sequence and the nucleic acid sequence capable of encoding an oleosin or a fragment thereof is capable of encoding a full length oleosin (See: FIGS. 2 c and 2 d ).
  • the nucleic acid sequence which upon transcription generates a RNA nucleic acid sequence that is complementary to a nucleic acid sequence encoding an oleosin is complementary to the full length oleosin nucleotide sequence, however only a fragment of a nucleic acid sequence encoding an oleosin is used. Preferably a fragment is selected which is capable of forming a hairpin.
  • the nucleic acid sequence encoding an oleosin is capable of encoding a full length oleosin and the RNA polynucleotide complementary to a nucleic acid sequence encoding an oleosin is complementary to only a fragment of the full length oleosin nucleotide sequence.
  • the fragment that is used is capable of forming a hairpin. The length of such a fragment may vary but will generally be 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotides in length (Thomas et al., (2001) Plant J. 25(4): 417-425).
  • nucleotide sequence encoding an oleosin or a fragment thereof where in said nucleotide sequence is complementary to a nucleic acid sequence that encodes a RNA polynucleotide complementary to a nucleic acid sequence encoding an oleosin mRNA or a fragment thereof is selected from SEQ ID NO:87 and 88.
  • a hairpin structure may be formed between the nucleic acid sequence encoding an RNA polynucleotide complementary to a nucleic acid sequence encoding an oleosin which is linked to a nucleic acid sequence encoding an oleosin.
  • the nucleic acid sequence which upon transcription generates a RNA nucleic acid sequence that is complementary to a nucleic acid sequence encoding an oleosin is separated by one of or more nucleotides from a nucleic acid sequence capable of encoding an oleosin (the sense sequence).
  • polynucleotide loop or “loop” have been defined previously herein and is shown schematically in FIG. 1 c.
  • said polynucleotide loop is 1 to 150 nucleotides in length. In a further preferred embodiment, said polynucleotide loop is 50 to 100 nucleotides in length and most preferably, said loop is 70 to 80 nucleotides in length. In a preferred embodiment, said polynucleotide loop is a poly A, poly U, poly C or poly G nucleotide chain. In a preferred embodiment, said poly A, poly U, poly C or poly G nucleotide chain is 2 to 150 nucleotides in length. In a further preferred embodiment, said poly A, poly U, poly C or poly G nucleotide chain is 10 to 150 nucleotides in length.
  • said poly A, poly U, poly C or poly G nucleotide chain is 50 to 100 nucleotides in length and most preferably, said poly A, poly U, poly C or poly G nucleotide chain is 20 to 80 nucleotides in length.
  • said polynucleotide loop comprises at least a poly A, poly U, poly C or poly G nucleotide chain wherein said poly A, poly U, poly C or poly G nucleotide chain comprises at least 2, 5, 10, 15, 20, 25, 30, 35 or 40 consecutive A, U, C or G nucleotide residues.
  • said loop comprises at least a poly A, poly U, poly C or poly G nucleotide chain wherein said poly A, poly U, poly C or poly G nucleotide chain comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the total length of said polynucleotide loop.
  • said polynucleotide loop comprises at least an (AC), (AU), (AG), (UC), (UG), (UA), (CU), (CG), (CA), (GU), (GA) or (GC) nucleotide chain wherein said (AC), (AU), (AG), (UC), (UG), (UA), (CU), (CG), (CA), (GU), (GA) or (GC) nucleotide chain comprises at least 1, 2, 5, 10, 15, 20, 25, 30, 35 or 40 consecutive (AC), (AU), (AG), (UC), (UG), (UA), (CU), (CG), (CA), (GU), (GA) or (GC) nucleotide residues.
  • said polynucleotide loop comprises at least a (AC), (AU), (AG), (UC), (UG), (UA), (CU), (CG), (CA), (GU), (GA) or (GC) nucleotide chain wherein said (AC), (AU), (AG), (UC), (UG), (UA), (CU), (CG), (CA), (GU), (GA) or (GC) nucleotide chain comprises at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the total length of said polynucleotide loop.
  • the polynucleotide loop is an intron
  • the intron is a plant intron.
  • Plant introns can vary widely in length, but approximately 2 ⁇ 3 of all plant introns are shorter than 150 nucleotides and the majority of introns fall in the range of 80 to 139 nucleotides in length (Simpson G G and Filipowicz W. (1996) Plant Mol Biol 32: 1-41.)
  • the minimal functional length of an intron has been determined to be approximately 70 nucleotides in higher plants (Goodall G J and Filipowics W. (1990) Plant Mol Biol 14: 727-733.).
  • the intron contains a classical splice site which consists of both a 5′ and 3′ splice site sequence.
  • the wider 5′ splice site consensus in higher plants is AG/GUAAGU.
  • the 5′ splice site comprises the /GU dinucleotide.
  • the 5′ splice site comprises a /GC dinucleotide.
  • the wider 3′ splice site consensus in plants is UGYAG/GU.
  • the 3′ splice site comprises the dinucleotide, AG/. (Simpson G G and Filipowicz W. (1996) Plant Mol Biol 32: 1-41.).
  • polynucleotide loop is an intron obtainable from a nucleotide sequence encoding an oleosin.
  • sequence of the polynucleotide loop is selected from SEQ ID NO:89-91.
  • the present invention provides a method to suppress expression of an oleosin protein in a plant comprising:
  • the present invention provides a method for preparing a plant seed derived product from plant seeds comprising:
  • the chimeric nucleic acid sequence is incorporated in a recombinant expression vector. Accordingly the present invention provides recombinant expression vectors suitable for the expression in a plant cell comprising a chimeric nucleic acid sequence comprising in the 5′ to 3′ direction of transcription:
  • the term “suitable for expression in the selected cell” means that the recombinant expression vector contains all nucleic acid sequences required to ensure expression in the selected cell. Accordingly, the recombinant expression vectors further contain regulatory nucleic acid sequences selected on the basis of the cell which is used for expression and ensuring initiation and termination of transcription operatively linked to the nucleic acid sequence encoding the modified oleosin. Nucleic acid sequences capable of controlling expression include promoters, enhancers, silencing elements, ribosome binding sites, Shine-Dalgarno sequences, introns and other expression elements.
  • “Operatively linked” is intended to mean that the nucleic acid sequences comprising the regulatory regions linked to the nucleic acid sequences encoding the anti-sense oleosin expression in the cell.
  • a typical nucleic acid construct comprises in the 5′ to 3′ direction a promoter region capable of directing expression, a coding region comprising the modified oleosin polypeptide and a termination region functional in the selected cell.
  • the selection of regulatory sequences will depend on the plant and the cell type in which the modified oleosin is expressed, and may influence the expression levels of the mRNA. Regulatory sequences are generally art-recognized and selected to direct expression of the modified oleosin in the cell.
  • Promoters functional in plant cells include constitutive promoters such as the 35S CaMV promoter (Rothstein et al., 1987 Gene 53: 153-161) the actin promoter (McElroy et al., 1990 Plant Cell 2: 163-171) and the ubiquitin promoter (European Patent Application 0 342 926).
  • Other promoters are specific to certain tissues or organs (for example, roots, leaves, flowers or seeds) or cell types (for example, leaf epidermal cells, mesophyll cells or root cortex cells) and or to certain stages of plant development. Timing of expression may be controlled by selecting an inducible promoter, for example the PR-a promoter described in U.S. Pat. No. 5,614,395. Selection of the promoter therefore depends on the desired location and timing of the accumulation of the desired polypeptide.
  • RNA polynucleotide complementary to a nucleic acid sequence encoding an oleosin mRNA or a fragment thereof expressed in a seed cell and seed specific promoters are utilized.
  • Seed specific promoters that may be used herein include for example the phaseolin promoter (Sengupta-Gopalan et al., 1985 Proc. Natl. Acad. Sci. USA: 82 3320-3324), and the Arabidopsis 18 kDa oleosin promoter (van Rooijen et al., 1992 Plant. Mol. Biol. 18: 1177-1179). New promoters useful in various plant cell types are constantly discovered.
  • the promoter is a constitutive promoter.
  • constitutive promoters include, but are not limited to, 35S CaMV promoter (Rothstein et al., 1987 Gene 53: 153-161) the actin promoter (McElroy et al., 1990 Plant Cell 2: 163-171) and the ubiquitin promoter (European Patent Application 0 342 926).
  • the promoter has the precise timing and tissue specificity of the oleosin gene to be suppressed. In the most preferred embodiment, the promoter from the oleosin gene to be suppressed is used.
  • Genetic elements capable of enhancing expression of the polypeptide may be included in the expression vectors.
  • these include for example, the untranslated leader sequences from viruses such as the AMV leader sequence (Jobling and Gehrke, 1987 Nature 325: 622-625) and the intron associated with the maize ubiquitin promoter (See: U.S. Pat. No. 5,504,200).
  • Transcriptional terminators are generally art recognized and besides serving as a signal for transcription termination serve as a protective element serving to extend the mRNA half-life (Guarneros et al., 1982 Proc. Natl. Acad. Sci. USA 79: 238-242).
  • the transcriptional terminator typically is from about 200 nucleotide to about 1000 nucleotides in length. Terminator sequences that may be used herein include for example, the nopaline synthase termination region (Bevan et al., 1983 Nucl. Acid. Res. 11: 369-385), the phaseolin terminator (van der Geest et al., 1994 Plant J.
  • the terminator for the octopine synthase gene of Agrobacterium tumefaciens or other similarly functioning elements Transcriptional terminators can be obtained as described by An (1987) Methods in Enzym. 153: 292. The selection of the transcriptional terminator may have an effect on the rate of transcription.
  • the recombinant expression vector further may contain a marker gene.
  • Marker genes that may be used in accordance with the present invention include all genes that allow the distinction of transformed cells from non-transformed cells including all selectable and screenable marker genes.
  • a marker may be a resistance marker such as an antibiotic resistance marker against for example kanamycin, ampicillin, G418, bleomycin hygromycin, chloramphenicol which allows selection of a trait by chemical means or a tolerance marker against for example a chemical agent such as the normally phytotoxic sugar mannose (Negrotto et al., 2000 Plant Cell Rep. 19: 798-803).
  • herbicide resistance markers may conveniently be used for example markers conferring resistance against glyphosate (U.S. Pat.
  • Recombinant expression vectors suitable for the introduction of nucleic acid sequences in plant cells include Agrobacterium and Rhizobium based vectors such as the Ti and Ri plasmids.
  • Agrobacterium based vectors typically carry at least one T-DNA border sequence and include vectors such pBIN 19 (Bevan, 1984 Nucl Acids Res. Vol. 12, 22:8711-8721) and other binary vector systems (for example: U.S. Pat. No. 4,940,838).
  • the nucleic acid sequence capable of controlling expression in plant cells permits expression in plant seed cells and the transformed plant is a plant capable of setting seed.
  • the promoter is a seed-preferred promoter. Accordingly the present invention provides a method to suppress expression of an oleosin protein in the seed of a plant comprising:
  • the recombinant expression vectors and chimeric nucleic acid sequences of the present invention may be prepared in accordance with methodologies well known to those skilled in the art of molecular biology. Such preparation will typically involve the bacterial species Escherichia coli as an intermediary cloning host.
  • the preparation of the E. coli vectors as well as the plant transformation vectors may be accomplished using commonly known techniques such as restriction digestion, ligation, gel ectrophoresis, DNA sequencing, the Polymerase Chain Reaction (PCR) and other methodologies.
  • PCR Polymerase Chain Reaction
  • a wide variety of cloning vectors is available to perform the necessary steps required to prepare a recombinant expression vector. Among the vectors with a replication system functional in E.
  • coli are vectors such as pBR322, the pUC series of vectors, the M13 mp series of vectors, pBluescript etc.
  • these cloning vectors contain a marker allowing selection of transformed cells.
  • Nucleic acid sequences may be introduced in these vectors, and the vectors may be introduced in E. coli grown in an appropriate medium.
  • Recombinant expression vectors may readily be recovered from cells upon harvesting and lysing of the cells. Further, general guidance with respect to the preparation of recombinant vectors may be found in, for example: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.
  • the recombinant expression vectors are introduced into the cell that is selected and the selected cells are grown to produce the modified oleosin protein in a progeny cell.
  • Methodologies to introduce recombinant expression vectors into a cell are well known to the art and vary depending on the cell type that is selected.
  • General techniques to transfer the recombinant expression vectors into the cell include electroporation; chemically mediated techniques, for example CaCl 2 mediated nucleic acid uptake; particle bombardment (biolistics); the use of naturally infective nucleic acid sequences for example virally derived nucleic acid sequences or when plant cells are used Agrobacterium or Rhizobium derived nucleic acid sequences; PEG mediated nucleic acid uptake, microinjection, and the use of silicone carbide whiskers (Kaeppler et al., 1990 Plant Cell Rep. 9:415-418) all of which may be used herein.
  • the recombinant expression vector into the cell may result in integration of its whole or partial uptake into host cell genome including the chromosomal DNA or the plastid genome.
  • the chimeric nucleic acid construct is introduced into the plant cell under nuclear genomic integration conditions. Under such conditions the chimeric nucleic acid sequence is stably integrated in the plant's genome.
  • the recombinant expression vector may not be integrated into the genome and replicate independently of the host cell's genomic DNA. Genomic integration of the nucleic acid sequence is typically used as it will allow for stable inheritance of the introduced nucleic acid sequences by subsequent generations of cells and the creation, plant lines.
  • Particular embodiments involve the use of plant cells.
  • Particular plant cells used herein include cells obtainable from Arabidopsis thaliana , Brazil nut ( Betholletia excelsa ); castor ( Riccinus coinnunis ); coconut ( Cocus nucifera ); coriander ( Coriandrum sativum ); cotton ( Gossypium spp.); groundnut ( Arachis hypogaea ); jojoba ( Simmondsia chinensis ); linseed/flax ( Linum usitatissimum ); maize ( Zea mays ); mustard ( Brassica spp.
  • Sinapis alba oil palm ( Elaeis guineeis ); olive ( Olea europaea ); rapeseed ( Brassica spp.); safflower ( Carthamus tinctorius ); soybean ( Glycine max ); squash ( Cucurbita maxima ); barley ( Hordeum vulgare ); wheat ( Traeticum aestivum ) and sunflower ( Helianthus annuus ).
  • Agrobacterium transformation generally involves the transfer of a binary vector (e.g. pBIN19) comprising the DNA of interest to an appropriate Agrobacterium strain (e.g. CIB542) by for example tri-parental mating with an E. coli strain carrying the recombinant binary vector and an E. coli strain carrying a helper plasmid capable of mobilization of the binary vector to the target Agrobacterium strain, or by DNA transformation of the Agrobacterium strain (Hofgen et al. Nucl. Acids.
  • a binary vector e.g. pBIN19
  • an appropriate Agrobacterium strain e.g. CIB542
  • transformation methodologies that may be used to transform dicotelydenous plant species include biolistics (Sanford, 1988 Trends in Biotechn. 6: 299-302); electroporation (Fromm et al., 1985 Proc. Natl. Acad. Sci. USA 82: 5824-5828); PEG mediated DNA uptake (Potrykus et al., 1985 Mol. Gen. Genetics 199: 169-177); microinjection (Reich et al., 1986 Bio/Techn. 4: 1001-1004) and silicone carbide whiskers (Kaeppler et al., 1990 Plant Cell Rep. 9: 415-418).
  • the exact transformation methodologies typically vary somewhat depending on the plant species that is used.
  • Arabidopsis safflower, or flax plant cells are used.
  • Safflower transformation has been described by Baker and Dyer (1996 Plant Cell Rep. 16: 106-110.
  • Flax transformation has been described by Dong J. and McHughen A. (Plant Cell Reports (1991) 10:555-560), Dong J. and McHughen A. (Plant Sciences (1993) 88:61-71) and Mlynarova et al. (Plant Cell Reports (1994) 13: 282-285).
  • Additional plant species specific transformation protocols may be found in: Biotechnology in Agriculture and Forestry 46: Transgenic Crops I (Y.P.S. Bajaj ed.), Springer-Verlag, New York (1999), and Biotechnology in Agriculture and Forestry 47: Transgenic Crops II (Y.P.S. Bajaj ed.), Springer-Verlag, New York (2001).
  • Monocotyledonous plant species may be transformed using a variety of methodologies including particle bombardment (Christou et al., 1991 Biotechn. 9: 957-962; Weeks et al., 1993 Plant Physiol. 102: 1077-1084; Gordon-Kamm et al., 1990 Plant Cell 2: 603-618) PEG mediated DNA uptake (EP 0 292 435; 0 392 225) or Agrobacterium -mediated transformation (Goto-Fumiyuki et al., 1999 Nature-Biotech. 17 (3):282-286).
  • particle bombardment Christou et al., 1991 Biotechn. 9: 957-962; Weeks et al., 1993 Plant Physiol. 102: 1077-1084; Gordon-Kamm et al., 1990 Plant Cell 2: 603-618
  • PEG mediated DNA uptake EP 0 292 435; 0 392 225
  • Agrobacterium -mediated transformation Goto-Fumiyuki et
  • Plastid transformation is described in U.S. Pat. Nos. 5,451,513; 5,545,817 and 5,545,818; and PCT Patent Applications 95/16783; 98/11235 and 00/39313.
  • Basic chloroplast transformation involves the introduction of cloned plastid DNA flanking a selectable marker together with the nucleic acid sequence of interest into a suitable target tissue using for example biolistics or protoplast transformation.
  • Selectable markers that may be used include for example the bacterial aadA gene (Svab et al., 1993 Proc. Natl. Acad. Sci. USA 90: 913-917).
  • Plastid promoters that may be used include for example the tobacco clpP gene promoter (PCT Patent Application 97/06250).
  • the invention chimeric nucleic acid contructs provided herein are directly transformed into the plastid genome.
  • Plastid transformation technology is described extensively in U.S. Pat. Nos. 5,451,513, 5,545,817, 5,545,818 and 5,576,198; in PCT application nos. WO 95/16783 and WO 97/32977; and in McBride et. al., 1994 Proc Natl Acad Sci USA 91: 7301-7305, the entire disclosures of all of which are hereby incorporated by reference.
  • plastid transformation is achieved via biolistics, first carried out in the unicellular green alga Chlamydomonas reinhardtii (Boynton et al., 1988 Science 240:1534-1537)) and then extended to Nicotiana tabacum (Svab et al., 1990 Proc Natl Acad Sci USA 87:8526-8530), combined with selection for cis-acting antibiotic resistance loci (spectinomycin or streptomycin resistance) or complementation of non-photosynthetic mutant phenotypes.
  • tobacco plastid transformation is carried out by particle bombardment of leaf or callus tissue, or polyethylene glycol (PEG)-mediated uptake of plasmid DNA by protoplasts, using cloned plastid DNA flanking a selectable antibiotic resistance marker.
  • PEG polyethylene glycol
  • 1 to 1.5 kb flanking regions termed targeting sequences, facilitate homologous recombination with the plastid genome and allow the replacement or modification of specific regions of the 156 kb tobacco plastid genome.
  • point mutations in the plastid 16S rDNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin can be utilized as selectable markers for transformation (Svab et al., 1990 Proc Natl Acad Sci USA 87:8526-8530; Staub et al., 1992 Plant Cell 4:39-45 the entire disclosures of which are hereby incorporated by reference), resulting in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers allows creation of a plastid targeting vector for introduction of foreign genes (Staub et al., 1993 EMBO J.
  • transformation frequency can be obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3′-adenyltransferase (Svab et al., 1993 Proc Natl Acad Sci USA 90: 913-917, the entire disclosure of which is hereby incorporated by reference).
  • plastid transformation of protoplasts from tobacco and the moss Physcomitrella can be attained using PEG-mediated DNA uptake (O'Neill et al., 1993 Plant J 3:729-738; Koop et al., 1996 Planta 199:193-201, the entire disclosures of which are hereby incorporated by reference).
  • a chimeric nucleic sequence construct is inserted into a plastid expression cassette including a promoter capable of expressing the construct in plant plastids.
  • a particular promoter capable of expression in a plant plastid is, for example, a promoter isolated from the 5′ flanking region upstream of the coding region of a plastid gene, which may come from the same or a different species, and the native product of which is typically found in a majority of plastid types including those present in non-green tissues.
  • Gene expression in plastids differs from nuclear gene expression and is related to gene expression in prokaryotes (Stern et al., 1997 Trends in Plant Sci 2:308-315, the entire disclosure of which is hereby incorporated by reference).
  • Plastid promoters generally contain the ⁇ 35 and ⁇ 10 elements typical of prokaryotic promoters, and some plastid promoters called PEP (plastid-encoded RNA polymerase) promoters are recognized by an E. coli -like RNA polymerase mostly encoded in the plastid genome, while other plastid promoters called NEP promoters are recognized by a nuclear-encoded RNA polymerase. Both types of plastid promoters are suitable for use herein.
  • PEP plastid-encoded RNA polymerase
  • plastid promoters examples include promoters of clpP genes such as the tobacco clpP gene promoter (WO 97/06250, the entire disclosure of which is hereby incorporated by reference) and the Arabidopsis clpP gene promoter (U.S. application Ser. No. 09/038,878, the entire disclosure of which is hereby incorporated by reference).
  • Another promoter capable of driving expression of a chimeric nucleic acid construct in plant plastids comes from the regulatory region of the plastid 16S ribosomal RNA operon (Harris et al., 1994 Microbiol Rev 58:700-754; Shinozaki et al., 1986 EMBO J.
  • a plastid expression cassette preferably further includes a plastid gene 3′ untranslated sequence (3′ UTR) operatively linked to a chimeric nucleic acid construct of the present invention.
  • the role of untranslated sequences is preferably to direct the 3′ processing of the transcribed RNA rather than termination of transcription.
  • An exemplary 3′ UTR is a plastid rps16 gene 3′ untranslated sequence, or the Arabidopsis plastid psbA gene 3′ untranslated sequence.
  • a plastid expression cassette includes a poly-G tract instead of a 3′ untranslated sequence.
  • a plastid expression cassette also preferably further includes a 5′ untranslated sequence (5′ UTR) functional in plant plastids, operatively linked to a chimeric nucleic acid construct provided herein.
  • a plastid expression cassette is contained in a plastid transformation vector, which preferably further includes flanking regions for integration into the plastid genome by homologous recombination.
  • the plastid transformation vector may optionally include at least one plastid origin of replication.
  • the present invention also encompasses a plant plastid transformed with such a plastid transformation vector, wherein the chimeric nucleic acid construct is expressible in the plant plastid.
  • a plant or plant cell including the progeny thereof, including this plant plastid.
  • the plant or plant cell, including the progeny thereof is homoplasmic for transgenic plastids.
  • promoters capable of driving expression of a chimeric nucleic acid construct in plant plastids include transactivator-regulated promoters, preferably heterologous with respect to the plant or to the subcellular organelle or component of the plant cell in which expression is effected.
  • the DNA molecule encoding the transactivator is inserted into an appropriate nuclear expression cassette which is transformed into the plant nuclear DNA.
  • the transactivator is targeted to plastids using a plastid transit peptide.
  • the transactivator and the transactivator-driven DNA molecule are brought together either by crossing a selected plastid-transformed line with and a transgenic line containing a DNA molecule encoding the transactivator supplemented with a plastid-targeting sequence and operably linked to a nuclear promoter, or by directly transforming a plastid transformation vector containing the desired DNA molecule into a transgenic line containing a chimeric nucleic acid construct encoding the transactivator supplemented with a plastid-targeting sequence operably linked to a nuclear promoter.
  • the nuclear promoter is an inducible promoter, in particular a chemically inducible embodiment
  • expression of the chimeric nucleic acid construct in the plastids of plants is activated by foliar application of a chemical inducer.
  • an inducible transactivator-mediated plastid expression system is preferably tightly regulatable, with no detectable expression prior to induction and exceptionally high expression and accumulation of protein following induction.
  • a particular transactivator is, for example, viral RNA polymerase.
  • Particular promoters of this type are promoters recognized by a single sub-unit RNA polymerase, such as the T7 gene 10 promoter, which is recognized by the bacteriophage T7 DNA-dependent RNA polymerase.
  • the gene encoding the T7 polymerase is preferably transformed into the nuclear genome and the T7 polymerase is targeted to the plastids using a plastid transit peptide.
  • Promoters suitable for nuclear expression of a gene for example a gene encoding a viral RNA polymerase such as the T7 polymerase, are described above and elsewhere in this application.
  • chimeric nucleic acid constructs in plastids can be constitutive or can be inducible, and such plastid expression can be also organ- or tissue-specific. Examples of various expression systems are extensively described in WO 98/11235, the entire disclosure of which is hereby incorporated by reference.
  • the present invention utilizes coupled expression in the nuclear genome of a chloroplast-targeted phage T7 RNA polymerase under the control of the chemically inducible PR-1a promoter, for example of the PR-1 promoter of tobacco, operably linked with a chloroplast reporter transgene regulated by T7 gene 10 promoter/terminator sequences, for example as described in as in U.S. Pat. No.
  • Cells may be harvested in accordance with methodologies known to the art. These methodologies are generally cell-type dependent and known to the skilled artisan. Where plants are employed they may be regenerated into mature plants using plant tissue culture techniques generally known to the skilled artisan. Seeds may be harvested from mature transformed plants and used to propagate the plant line. Plants may also be crossed and in this manner, contemplated herein is the breeding of cells lines and transgenic plants that vary in genetic background.
  • plant genomes may comprise one or more nucleotide sequences encoding an oleosin, typically each varying somewhat in nucleic acid sequence. It may be desirable to simultaneously suppress the expression of a plurality of oleosins. Accordingly a plurality of chimeric sequences may be prepared, each designed to suppress expression of a different oleosin nucleotide sequence. In accordance herewith separate vectors comprising such chimeric nucleic acid sequences may simulataneously be introduced into a plant cell.
  • a single vector comprising a plurality of chimeric nucleic acid sequences, each chimeric sequence comprising (i) a nucleic acid sequence capable of directing transcription in a plant cell; (ii) a nucleic acid sequence which upon transcription generates a RNA nucleic acid sequence that is complementary to a nucleic acid sequence encoding an oleosin and (iii) a nucleic acid sequence encoding an oleosin may be introduced into a plant cell (see FIG. 2 b, g, h, i ).
  • such a plant upon having prepared a plant using one chimeric nucleic acid sequence in accordance herewith, such a plant may be transformed with one or more additional chimeric nucleic acid sequences each targeting a different endogenous plant oleosin.
  • the present invention also provides plants and plant seeds in which oleosin gene expression has been suppressed comprising in the 5′ to 3′ direction of transcription as operably linked components:
  • the present invention also includes oil bodies comprising a modified oleosin profile of the invention.
  • seed obtained in accordance with the present invention may be used to prepare a seed derived product.
  • Seed derived products that may be prepared in accordance with the present invention include products for human and animal use, including food and feed products and personal care products.
  • the plant seed derived product may be prepared using any standard commercial processing practices for seed. Whole seeds or crushed seeds may be used to prepare the seed derived products, for example food products. Alternatively seed fractions are prepared which then are used to prepare the seed derived product. Preferred methods that may be used in accordance herewith include solvent extraction, such as extraction by hexane and the application of mechanical force, for example pressing, grinding or milling. Typically, these processes result in the separation of the seed oil fraction from the protein, also termed the meal, fraction. The isolated meal and oil fraction may both be used for further processing in food or feed products or personal care products.
  • Seed derived products for human consumption that may be prepared in accordance with the present invention include any food product including any health food that is capable of imparting health benefits.
  • Beverages that may be prepared from seed products prepared in accordance with the present invention include any beverage in dry powdered or liquid form, for example any fruit juice, fresh frozen or canned concentrate, flavored drinks as well as adult and infant formulas.
  • Further products include products prepared from a non-dairy milk, such as soy milk. These products include whole milk, skim milk, ice cream, yoghurt and the like.
  • Animal feed products that may be prepared using seed prepared in accordance with the present invention include products intended feed products, including products for use in fish and shrimp aquaculture, and pet food products, intended for feeding to dogs, cats, birds, reptiles, rodents and the like.
  • Personal care products that may be prepared using a seed derived product prepared in accordance with the present invention include any cosmetic product for human use, including soaps, skin creams facial creams, face masks, skin cleanser, tooth paste, lipstick, perfumes, make-up, foundation, blusher, mascara, eyeshadow, sunscreen lotions, hair conditioner, and hair colouring.
  • the exact seed processing conditions as well as the plant seed derived product preparation methodology, employed will vary depending on the plant species as well as on the desired plant seed derived product the seed is processed into.
  • the exact processing conditions of the seed or the preparation techniques for the seed derived employed are considered to be immaterial to the present invention.
  • the present invention provides a method to alter the composition of plants, notably plant seeds.
  • seeds may be prepared in which the lipid content is reduced, whereas the protein content within the seed is increased relative to seed obtained from wild type plant seeds.
  • the present invention also provides a method to increase the protein content in plants seeds comprising:
  • the present invention also provides a method to decrease the lipid content in plants seeds comprising:
  • the seeds thus obtained may be used to prepare a plant seed derived product.
  • the plant seeds with a modified oleosin protein profile within the seed have an increase in the total protein content of said plants seeds.
  • this increase in protein results after the introduction of the chimeric nucleic acid sequence into the plant cell and regeneration of a mature plant.
  • the increase in total protein content of the plant seeds with a modified oleosin profile is increased to a level from about 5% to about 30% relative to the total protein content of the plant seeds from wild type plants with unaltered protein levels.
  • the total protein content of the plant seeds with a modified oleosin profile is increased a level 15% to 30% relative to the total protein content of the plant seeds from wild type plants with unaltered protein levels and most preferably, the total protein content of the plant seeds with a modified oleosin profile is increased to a level 20% to 30% compared to the total protein content of the plant seeds from wild type plants not comprising the chimeric nucleic acid sequence of the present invention.
  • Techniques to determine the total protein content of plant seeds include using BCA protein assay reagent (Pierce, Rockford, Ill.) and further described in Example 5 of the present application. The techniques listed above may be performed on either a total seed extract or on the oil body fraction.
  • the plant seeds with a modified oleosin protein profile within the seed have a decrease in the lipid content in said plants seeds.
  • this decrease in lipid content results after the introduction of the chimeric nucleic acid sequence into the plant cell and regeneration of a mature plant.
  • the decrease in lipid content of the plant seeds with a modified oleosin profile is decreased to a level from about 1% to about 20% relative to the lipid content of the plant seeds from wild type plants with unaltered protein levels.
  • the lipid content of the plant seeds with a modified oleosin profile is decreased a level 10% to 20% relative to the lipid content of the plant seeds from wild type plants with unaltered protein levels and most preferably, the lipid content of the plant seeds with a modified oleosin profile is increased to a level 15% to 20% compared to the lipid content of the plant seeds from wild type plants not comprising the chimeric nucleic acid sequence of the present invention.
  • Techniques to determine the lipid content of plant seeds include are described in Bligh and Dyer (1959. Can. J. Med. Sci. 37:911-917) and further described in Example 5 of the present application.
  • One seed fraction that may be obtained in accordance with the present invention in order to prepare a seed derived product is the oil body fraction. Accordingly, in another aspect of the present invention, the oil body fraction may be obtained using for example methods as disclosed in PCT 98/53698 and the oil body fraction may be used to prepare food, feed or personal care products.
  • the present invention provides a composition comprising oil bodies with a modified oleosin profile isolated from plant seeds. Accordingly, the present invention provides a composition comprising oil bodies with a modified oleosin profile isolated from plant seeds.
  • the oil bodies with a modified oleosin profile are preferably prepared by a process comprising:
  • Seed grinding may be accomplished by any comminuting process resulting in a substantial disruption of the seed cell membrane and cell walls without compromising the structural integrity of the oil bodies present in the seed cell.
  • Suitable grinding processes in this regard include mechanical pressing and milling of the seed.
  • Wet milling processes such as described for cotton (Lawhon et al., 1977 J. Am. Oil Chem. Soc. 63: 533-534) and soybean (U.S. Pat. No. 3,971,856; Carter et al., 1974 J. Am. Oil Chem. Soc. 51: 137-141) are particularly useful in this regard.
  • Suitable milling equipment capable of industrial scale seed milling include colloid mills, disc mills, pin mills, orbital mills, IKA mills and industrial scale homogenizers. The selection of the milling equipment will depend on the seed, which is selected, as well as the throughput requirement.
  • Solid contaminants such as seed hulls, fibrous materials, undissolved carbohydrates, proteins and other insoluble contaminants are subsequently preferably removed from the ground seed fraction using size exclusion based methodologies such as filtering or gravitational based methods such as a centrifugation based separation process.
  • Size exclusion based methodologies such as filtering or gravitational based methods such as a centrifugation based separation process.
  • Centrifugation may be accomplished using for example a decantation centrifuge such as a HASCO 200 2-phase decantation centrifuge or an NX310B (Alpha Laval). Operating conditions are selected such that a substantial portion of the insoluble contaminants and sediments and may be separated from the soluble fraction.
  • Gravitational based methods as well as size exclusion based technologies may be used. Gravitational based methods that may be used include centrifugation using for example a tubular bowl centrifuge such as a Sharples AS-16 or AS-46 (Alpha Laval), a disc stack centrifuge or a hydrocyclone, or separation of the phases under natural gravitation. Size exclusion methodologies that may be used include membrane ultra filtration and crossflow microfiltration.
  • Separation of solids and separation of the oil body phase from the aqueous phase may also be carried out concomitantly using gravity based separation methods or size exclusion based methods.
  • the oil body preparations obtained at this stage in the process are generally relatively crude and depending on the application of the oil bodies, it may be desirable to remove additional contaminants. Any process capable of removing additional seed contaminants may be used in this regard. Conveniently the removal of these contaminants from the oil body preparation may be accomplished by resuspending the oil body preparation in an aqueous phase and re-centrifuging the resuspended fraction. The resuspension conditions selected may vary depending on the desired purity of the oil body fractions. The oil bodies may be resuspended one or more times depending on the desired purity and the ionic strength, pH and temperature may all be varied. Analytical techniques may be used to monitor the removal of contaminants. For example SDS gel electrophoresis may be employed to monitor the removal of seed proteins.
  • the entire oil body isolation process may be performed in a batch wise fashion or continuous flow.
  • industrial scale continuous flow processes are utilized.
  • the oil bodies isolated from a plant with a modified oleosin profile are larger then oil bodies found in the wild type plant. For the purpose of this application, this increase in size results after the introduction of the chimeric nucleic acid sequence into the plant cell and regeneration of a mature plant.
  • the size of the oil bodies with a modified oleosin profile are increased to a level from about 1, 2, 3, 4, 5, 6, 7, 8, 9 to about 10 times compared to the oil bodies from wild type plants with unaltered protein levels.
  • the size of the oil bodies with a modified oleosin profile is increased to a level that is 2 times, more preferably 5 to 10 times, compared to the oil bodies from wild type plants with unaltered protein levels.
  • the size of the oil bodies with a modified oleosin profile is increased to a level 8 to 10 times compared to the oil bodies from wild type plants not comprising the chimeric nucleic acid sequence of the present invention.
  • Techniques to determine the size of the oil bodies in vivo include confocal microscopy. This technique allows the examination of whole mount, embryo sections and provides a more accurate size comparison between Arabidopsis lines. Embryos can be isolated from mature seeds and neutral lipids stained with Nile Red. Triacylglycerols represent the vast majority of neutral lipids in most oil seeds hence oil bodies are selectively stained by Nile Red. Examples of protocols can be found in Paddock et al. Methods in Molecular Biology, vol 122.
  • the plant seeds with a modified oleosin protein profile within the seed have decrease in the phospholipid accumulation in said plants seeds.
  • this decrease in phospholipid accumulation results after the introduction of the chimeric nucleic acid sequence into the plant cell and regeneration of a mature plant.
  • the decrease in phospholipid accumulation of the plant seeds with a modified oleosin profile is decreased to a level from about 5% to about 40% relative to the phospholipid accumulation of the plant seeds from wild type plants with unaltered protein levels.
  • the phospholipid accumulation of the plant seeds with a modified oleosin profile is decreased a level 20% to 40% relative to the phospholipid accumulation of the plant seeds from wild type plants with unaltered protein levels and most preferably, the phospholipid accumulation of the plant seeds with a modified oleosin profile is increased to a level 30% to 40% compared to the phospholipid accumulation of the plant seeds from wild type plants not comprising the chimeric nucleic acid sequence of the present invention.
  • Techniques to determine the phospholipid accumulation of plant seeds include are described in Vance and Russel, 1990 (J. Lipid Res 31:1491-1501.) and further described in Example 6 of the present application.
  • the present invention further provides a method to modulate the oleosin constituents in a plant seed.
  • a nucleic acid sequence encoding an oleosin may be introduced in such a plant line.
  • the nucleic acid sequence encoding such an oleosin may be obtained from a different plant species.
  • the present invention further describes a method to increase accumulation of recombinant proteins in the surface of oil bodies.
  • the method is based on the suppression of endogenous oleosins with concomitant expression of a recombinant oleosin.
  • the modified oil bodies may contain higher amounts of recombinant oleosins.
  • said recombinant oleosin is covalently linked to a second recombinant protein to form a chimeric protein as disclosed in WO 93/21320 and related applications which are incorporated by reference in its entirety.
  • the use of a recombinant oleosin protein as a carrier or targeting means provides a simple mechanism to recover proteins.
  • the chimeric protein associated with the oil body may be separated away from the bulk of cellular components in a single step by isolation of the oil body fraction using for example centrifugation size exclusion or floatation.
  • the invention contemplates the use of heterologous proteins, including enzymes, therapeutic proteins, diagnostic proteins and the like fused to a recombinant oleosin and associated with oil bodies. Association of the protein with the oil body allows subsequent recovery of the protein by simple means (centrifugation and floatation). Accordingly the present invention further includes a method for the preparing a plant seed derived product from plants seeds comprising:
  • the oil bodies with a suppressed level of endogenous oleosins with concomitant expression of a recombinant oleosin have an increased density or expression level of recombinant oleosins on the surface of said oil bodies when compared to the expression level of a recombinant oleosin in a wild type plant where the endogenous oleosins are not suppressed.
  • the expression level of the recombinant oleosin on an oil body from a plant with suppressed levels of endogenous oleosins is increased to a level from about 1% to about 20% when compared to the expression level of a recombinant oleosin on an oil body from a plant where the endogenous oleosins are not suppressed.
  • the expression level of the recombinant oleosin on an oil body from a plant with suppressed levels of endogenous oleosins is increased to a level from about 10% to 20% when compared to the expression level of a recombinant oleosin on an oil body from a plant where the endogenous oleosins are not suppressed and most preferably, the expression level of the recombinant oleosin on an oil body from a plant with suppressed levels of endogenous oleosins is increased to a level from about 15% to 20% when compared to the expression level of a recombinant oleosin on an oil body from a plant where the endogenous oleosins are not suppressed.
  • the present invention further describes uses of oil bodies with an increased accumulation of recombinant oleosin covalently linked to a second recombinant protein on the surface of oil bodies.
  • the oil body with an increased accumulation of recombinant fusion proteins in the surface of the oil body can be used as an affinity matrix (see WO 98/27115 and related applications, all which are incorporated herein by reference).
  • affinity matrix see WO 98/27115 and related applications, all which are incorporated herein by reference.
  • it was found that oil bodies and their associated proteins can be used as affinity matrices for the separation of a wide variety of target molecules such as proteins, carbohydrates, lipids, organic molecules, nucleic acids, metals, cells and cell fractions from a sample.
  • a method for the separation of a target molecule from a sample comprising: 1) contacting (i) oil bodies that can associate with the target molecule through a ligand or second recombinant protein which is covalently attached to a recombinant oleosin with (ii) a sample containing the target molecule; and 2) separating the oil bodies associated with the target molecule from the sample.
  • the oil bodies and the sample containing the target molecule are brought into contact in a manner sufficient to allow the oil bodies to associate with the target.
  • oil bodies are mixed with the target. If desired, the target molecule may be further separated from the oil bodies.
  • the ligand fused to the oil body protein may be hirudin and can be used to purify thrombin.
  • the ligand fused to the oil body protein may be metallothionein and can be used to separate cadmium from a sample.
  • the ligand fused to the oil body protein may be protein A and can be used to separate immunoglobulins.
  • the ligand fused to the oil body protein may be cellulose binding protein and can be used to separate cellulose from a sample.
  • the present invention further describes uses of oil bodies with an increased accumulation of recombinant oleosin covalently linked to a second recombinant protein on the surface of oil bodies.
  • the low endogenous oleosin background could allow the display of antigenic polypeptides on the surface of oil bodies, improving their use as an adjuvant in a immunogenic formulation or a vaccine or as an immunogenic formulation (see WO 01/95934 and related applications and U.S. Pat. No. 6,761,914 and related patents and patent applications, all which are incorporated herein by reference).
  • a recombinant oleosin is covalently linked to the antigen or second recombinant protein (as disclosed in WO 93/21320 and related applications which are incorporated by reference in their entirely) which can be physically associated with the oil bodies in the vaccine or immunogenic formulation.
  • the vaccines or immunogenic formulations of the present invention can be used to elicit an immune response against any antigen using any route of administration including transdermal or through the mucosa.
  • the Atol1 cDNA was amplified using the forward primer NTD (5′-TATTAAGCTTCCATGGCCGATACTGCTAGAGG-3′) (SEQ ID NO:92) containing HindIII and NcoI restriction sites (underlined) and the reverse primer CTR (5′-AGCCATACTAGTAGTGTGTTGACCACCACCACGAG-3′) (SEQ ID NO:93) containing the SpeI restriction site (underlined) using Atol1 cDNA (SEQ ID NO:94) as a template.
  • the PCR product was purified and inserted in the vector pSBS2090, under control of the phaseolin promoter/terminator (Slightom et al., 1983 Proc. Natl. Acad. Sci.
  • This vector was previously digested with the restriction enzyme SwaI ( FIG. 3 b ).
  • the PCR product can be inserted in a direct or inverted orientation because the enzyme SwaI generates blunt ends.
  • the plasmids containing Atol1 cDNA in the inverse orientation was screened with the enzyme NcoI.
  • a vector containing Atol1 cDNA in the inverse orientation digested with NcoI releases a DNA fragment with 55 lbp ( FIG. 3 c ).
  • the hairpin cassette was constructed following the same scheme described for the antisense. The only difference was that during the ligation reaction the amount of the PCR product was increased to allow the insertion of two inverted repeats of Atol1 cDNA in pSBS2090. The number of copies of Atol1 inserted was analyzed through digestion with Xba and HindIII. Each copy of Atol1 was 555 bp in length and a dimer would have 1110 bp. To check the orientation of the two copies, digestion with NcoI was performed. If the vector contained an inverted repeat of Atol1 cDNA, digestion with NcoI would release a fragment with 1054 bp ( FIG. 3 d ).
  • the hairpin+intron cassette was constructed by inserting an intron in the hairpin cassette.
  • the unique intron of Atol1 was amplified using the forward primer IntronD (5′-TTTTACTAGTGATTTACAAtTAAGCACACATTTATC-3′) (SEQ ID NO:95) containing SpeI restriction site (underlined) and the reverse primer IntronR (5′-CTGTACTAGTTCTCCCGTTGCGTACCTATTCAC-3′) (SEQ ID NO:96) containing the SpeI restriction site (underlined) using an Atol1 genomic clone as template.
  • the PCR product was purified and digested with SpeI ( FIG. 4 d ).
  • the hairpin+intron cassette was sub-cloned in the plasmid pUC19 (New England Biolabs Inc.) in the Kpn and BamHI restriction sites ( FIG. 4 c ).
  • the resulting vector was digested with SpeI restriction enzyme between the inverted repeats of Atol1 cDNA.
  • the Atol1 intron was inserted between the repeats ( FIG. 4 e ). The orientation of the insertion was verified through PCR.
  • the antisense (SEQ ID NO:97), hairpin (SEQ ID NO:98) and hairpin+intron cassettes (SEQ ID NO:99) are inserted in the binary vector pSBS3000 in the sites KpnI and BamHI ( FIG. 5 ), creating the vectors pSBS3000-antisense, pSBS3000-hairpin and pSBS3000-hairpin+intron.
  • the binary vectors pSBS3000-antisense, pSBS3000-hairpin and pSBS3000-hairpin+intron were individually inserted in Agrobacterium EHA101 (Hood, E. E. et al. 1986. Journal of Bacteriology 168:1291-1301) by electroporation method.
  • the transformed Agrobacterium lines containing the binary vector were selected using spectinomycin resistance (“SpecR” in FIG. 5 e ). One line of Agrobacterium was selected for each construct.
  • the plants were irrigated at 2-3 day interval and fertilized weekly with 1% of Peters 20-20-20. When stems reach about 2 cm in height, the primary bolts were cut to encourage the growth of secondary and tertiary bolts. Four to five days after cutting the primary bolts, the plants were ready to be infected with Agrobacterium.
  • the Agrobacterium lines were individually inoculated in 500 ml of LB media and grown until they reached an optical density of 0.8 at 600 nm. The cultures were centrifuged to precipitate the bacteria that was subsequently suspended in a solution containing 5% of sucrose and 0.05% of the surfactant Silwet L-77 (Lehle Seeds).
  • the pots with Arabidopsis plants were inverted in the solution for 20 seconds.
  • the pots were subsequently covered with a transparent plastic dome for 24 hours to maintain higher humidity.
  • the plants were allowed to grow to maturity and seeds (untransformed and transformed) were harvested.
  • the putative transformed seeds were sterilized with a quick wash of 70% ethanol and a treatment in 20% commercial bleach for 15 min.
  • the bleach solution was removed by rinsing seeds four times with water.
  • About 1000 sterilized seeds were mixed with 0.6% top agar and evenly spread on a half strength MS plate (Murashige and Skoog 1962. Physiologia Plantarum 15:473-497) containing 1% sucrose and 80 ⁇ M of the herbicide phosphinothricin (PPT) DL.
  • PPT herbicide phosphinothricin
  • putative transgenic seedlings were green and growing whereas untransformed seedlings were dead. After the roots have established the putative transgenic seedlings were individually transferred to pots (the individual plants were irrigated in 3 day intervals and fertilized with 1% Peters 20-20-20 in 5 day intervals and allowed to grow to maturity). The pots were covered with a transparent plastic dome for three days to protect the sensitive seedlings. After 7 days the seedlings were covered with a seed collector from Lehle Seeds to prevent seed loss due to scattering. Seeds from these transgenic plants are harvested individually and ready for analysis.
  • Atol1 in seeds recovered from the selected plants was analyzed by SDS-PAGE of the oil body fraction.
  • Oil bodies from these seeds were obtained using the method reported by van Rooijen & Moloney, (1995) Biotechnology (N.Y.) 13, 72-77 with the following modifications. Briefly, 10 to 20 mg of dry mature seeds were ground inside a 1.7 ml microfuge tube with 0.4 ml of oil body extraction buffer (50 mM Tris-HCl pH 7.5 with 0.4M of sucrose and 0.5M of NaCl). The extract was centrifuged for 15 min at 10,000 g at room temperature (RT). After centrifugation the fat pad containing the oil bodies was removed from the aqueous phase and transferred to another microfuge tube.
  • oil body extraction buffer 50 mM Tris-HCl pH 7.5 with 0.4M of sucrose and 0.5M of NaCl
  • the oil bodies were resuspended in 0.4 ml of high stringency urea buffer (8M Urea in 100 mM Na-Carbonate buffer pH 8.0). The sample was centrifuged for 15 min at 10,000 g at 4° C. and the undernatant removed. The oil bodies were finally suspended in 0.1 ml of water. The presence of Atol1 in oil body fractions was detected by loading 20 ⁇ l of oil body fraction in SDS-PAGE 15% and staining with Coomassie blue ( FIG. 6 ).
  • Atol1 A decrease in the level of Atol1 was observed in the oil body fraction for all of the cassettes (Antisense—lane 3, Hairpin—lane 2 and Hairpin-Loop—lane 1) with a higher level of suppression in the Hairpin-Loop cassette when compared to the wild type oil body fraction.
  • Morphological analysis of oil bodies can be performed in vivo using dark field confocal microscopy or in vitro using bright field conventional microscopy.
  • Mature embryos were isolated according the method established by Perry and Wang (2003 Biotechniques 35:278-281).
  • confocal microscopy dark field microscopy
  • isolated embryos were infiltrated with an aqueous solution of 10 ⁇ g/ml Nile red (Molecular Probes) for neutral lipid staining (Greenspan et al., 1985. J. Cell Biol. 100:965-973).
  • the sections were stained using periodic acid-Schiff's reaction and counterstained with an alkaline toluidine blue O solution (Yeung, 1990. Stain Technol. 65:45-47.).
  • the resulting micrographs show the embryo cell structure where the protein bodies are stained in purple and the oil bodies are present as hollow structures ( FIG. 8 ).
  • oil bodies were present as small units (1 ⁇ m) ( FIG. 8A ).
  • the oil bodies obtained from transgenic plants transformed with pSBS3000-hairpin+intron and pSBS3000-hairpin were considerably more heterogeneous in size ranging from sizes similar to that of the wild type to large (up to 6 ⁇ m) and protein bodies were very irregular ( FIG. 8B ).
  • the first extraction was performed with 5.8 ml of MeOH:CHCl 3 :H 2 O (2:2:1.8 [v/v]) and the second and third extractions are performed with 2.0 ml of MeOH:CHCl 3 :H 2 O (1:2:0.8 [v/v]).
  • the lipid fractions were collected and the solvents were evaporated under a nitrogen environment. Total lipids were quantified by gravimetric analysis.
  • Seed protein accumulation in plants transformed with Hairpin+intron cassette was analyzed using the BCA protein assay reagent (Pierce, Rockford, Ill.). Total seed proteins were extracted from 50 mg of seeds homogenized in 1.5 ml of protein extraction buffer (2% SDS, 5 mM EDTA, 50 mM Tris-HCl, pH 6.8). The homogenates were placed in boiling water for 5 minutes and centrifuged at full speed for 10 minutes. The upper phase was removed and the debris was washed two times with 0.5 ml of extraction buffer. The fractions were pooled and the amount of protein was measured with the BCA protein assay reagent.
  • the insoluble fraction from the ethanol extraction was suspended in 0.2 ml of 0.2M KOH and incubated 95° C. for 1 h. The solution was neutralized with 35 ml of 1M acetic acid and centrifuged for 5 minutes at full speed. The supernatant was used for starch quantification. Sucrose and starch were determined using kits from Sigma-Aldrich (Oakville, ON).
  • Atol1 isoform of oleosin resulted in a decrease in lipid accumulation accompanied by an increase in protein content. While the ratio of lipid and protein content changed, the total weight of protein and oil remained constant. Analysis of sucrose and starch content revealed no significant difference in the accumulation of these carbohydrates. (Table 2).
  • Oil bodies were isolated from seeds and suspended in water. Total lipids were extracted from aliquots oil body suspension with methanol and chloroform through the method described by Bligh and Dyer (1959). Three extractions were performed and the solvents were evaporated under nitrogen environment. Total lipids were quantified through gravimetry. To measure the amount of proteins, the oil body suspension was boiled in the presence of 2% of SDS and centrifuged. The undernatant, containing the oil body proteins, was collected and used in BCA protein assays. The percentage of lipids and proteins was calculated considering the sum of both masses as the total mass of oil bodies. The oil bodies from transgenic plants transformed with pSBS3000-hairpin+intron cassette contained less protein, or a lower oleosin-to-TAG ratio, than those from the wild type plants.
  • the composition of lipids in the oil body fraction was evaluated through thin layer chromatography.
  • Total lipids extracted from oil bodies were loaded in similar amounts on a silica-gel plate.
  • the plate was half-developed with the mixture chloroform-methanol-acetic acid-formic acid-water (70:30:12:4:2 [v/v]) to resolve phospholipids and fully developed with the mixture hexane-diethyl ether-acetic acid (65:35:2 [v/v]) to allow the separation of neutral lipids (Vance and Russel, 1990).
  • Lipids were visualized through charring in the presence of cupric sulphate. The majority of lipid was composed of TAG with smaller amounts of cholesterol ester and other neutral lipids.
  • a gene coding for a recombinant oleosin was introduced in the Hairpin-Loop line.
  • an oleosin from Maize (MaizeOle1, accession number U13701) was selected to restore the function because it is phylogenetically distant from Atol1 (Huang, 1996; Lee et al., 1994).
  • FIG. 10A The Hairpin-Loop Arabidopsis plant ( FIG. 10A ) was manually crossed with MaizOl, an Arabidopsis line expressing MaizeOle1 under control of the linin seed specific promoter ( FIG. 10B ).
  • MaizeOle1 has distinct molecular weight (15.8 kDa) when compared to Arabidopsis oleosins. We used this property to analyze the progeny of the crossed lines.
  • the lines showing the presence of Maize oleosin and suppression of Atol1 were selected and propagated for two more generations. A homozygous line was obtained and the seeds were analyzed in a confocal microscope ( FIG. 10C ).
  • Oil bodies in this line did not display the phenotype found in the Hairpin-Loop line. Such oil bodies were still larger than wild-type ones but uniform in size, like those found in the Antisense line. This result indicates that the size of the oil bodies is controlled by the level of oleosin protein.
  • FIGS. 11E and 11F When seeds were germinated in MS media with or without sucrose kept in the dark ( FIGS. 11E and 11F ) or media without sucrose exposed to light ( FIG. 11C ).
  • the delay in seed germination could be reverted when seeds were sown in media with sucrose and exposed to light ( FIG. 11D ) or when seeds were sown on moistened paper and submitted to stratification for 3 days ( FIG. 11B ).
  • FIGS. 12F and G After germination the development of oleosin-suppressed seedlings was comparable to wild type ( FIGS. 12F and G). Usually oil bodies are consumed during the first days after imbibition. Our experiments demonstrated that, two days after imbibition, the oil bodies were still present in the boundaries of the cells as small units. After four days they are scarcely found and have completely disappeared at the fifth day ( FIGS. 12A and 12B ). In oleosin suppressed plants oil bodies assume different behaviour. Two days after germination they are found as large structures with about 10 ⁇ m in the cytoplasm. Four days after imbibition the number and size and of oil bodies decrease although large structures are still present in some cells.
  • FIGS. 12C to 12E Six days after germination some oil bodies can still be found as large structures but after that they completely disappear ( FIGS. 12C to 12E ). The slower mobilization of TAGs does not seem to affect post germination growth ( FIGS. 12F and 12G ). Although some seedlings seem to be smaller in the Hairpin-Loop line this is most likely due to a delay in germination.
  • SEQ ID NO:1 to 84 set forth known oleosin sequences which are described in Table 1.
  • SEQ ID NO:85 and 86 set forth the nucleic acid sequences of the antisense sequences.
  • SEQ ID NO:87 and 88 set forth nucleic acid sequences of the sense sequences.
  • SEQ ID NO:89 to 91 set forth the nucleic acid sequences of the loop sequences.
  • SEQ ID NO:92 sets forth the nucleotide sequence of the forward primer NTD which is complementary to the 5′ region of the Atol 1 cDNA clone and is designed to add HindIII and NcoI restriction sites site to the 5′ region facilitate subsequent ligation.
  • SEQ ID NO:93 sets forth the nucleotide sequence of the reverse primer CTR which is complementary to 3′ region of the C-terminal domain of Atol 1 cDNA and is designed to add a SpeI site to the 3′ region facilitate subsequent ligation.
  • SEQ ID NO:94 sets forth the nucleotide sequence of the Atol 1 cDNA sequence.
  • SEQ ID NO:95 sets forth the nucleotide sequence of the forward primer Intron D which is complementary to the 5′ region of the 5′ border of the intron of Atol 1 (including the 3′ region of exon 1) and is designed to add SpeI restriction sites site to the 5′ region facilitate subsequent ligation.
  • SEQ ID NO:96 sets forth the nucleotide sequence of the reverse primer Intron R which is complementary to the 3′ region of the 3′ border of the intron of Atol 1 (including the 5′ region of exon 2) and is designed to add SpeI restriction sites site to the 3′ region facilitate subsequent ligation.
  • SEQ ID NO:97 sets forth the nucleotide sequence of the antisense cassette as described in Example 1.
  • SEQ ID NO:98 sets forth the nucleotide sequence of the hairpin construct as described in Example 1.
  • SEQ ID NO:99 sets forth the nucleotide sequence of the hairpin and intron cassette as described in Example 1.

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US20050244050A1 (en) * 2002-04-25 2005-11-03 Toshio Nomura Image data creation device, image data reproduction device, and image data recording medium
US20110006221A1 (en) * 2008-02-28 2011-01-13 Toyota Jidosha Kabushiki Kaisha Method for evaluating oil-and-fat amount in seed and method for screening for plant exhibiting varied level of oil-and-fat content
US8173435B2 (en) 2008-02-28 2012-05-08 Toyota Jidosha Kabushiki Kaisha Method for evaluating oil-and-fat amount in seed and method for screening for plant exhibiting varied level of oil-and-fat content
US20110123448A1 (en) * 2009-11-25 2011-05-26 China Medical University Oil body carriers, uses in target therapy and/or detection of the same, and fusion proteins comprised therein

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CA2582944A1 (en) 2006-04-13
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